Molecular genetics, evolution and phylogeography of mammal species

Chimpanzees

Our cover article in the journal Science (Publication 146) was the first study of the genetics of wild chimpanzees and received worldwide media coverage and scientific scrutiny. It was especially noteworthy for its innovative use of noninvasive genotyping and pioneering demonstration of the power of nuclear microsatellite-based population genetic analyses for animals other than man. We have now undertaken detailed genetic studies of pedigree relationships and population structure of entire habituated chimpanzee social communities at Gombe, Tanzania, and in the Tai forest of Cote d'Ivorie. Other papers concern paternity exclusion, pedigree relations and population structure in captive and wild chimpanzees (Publications 120, 139, 149).

Graduate student, Philip Morin and I focused initially on the Gombe community in Tanzania, taking advantage of 30 years of observational data gathered by Jane Goodall. Using shed hair collected from night nests, we used mitochondrial sequences and hypervariable nuclear markers called microsatellites to make a number of remarkable discoveries that were published in 1994. We found that the existing taxonomic treatment of chimpanzees, universally accepted for 40 years, was wrong; that female gene flow can be detected over distances of up to 600 km; and that within social groups one can establish pedigree relationships, lifetime reproductive success, and innate variability. Graduate student, Pascal Gagneux, extended our investigation to West Africa, and to the chimpanzees of the Tai forest, Cote d'Ivorie, in particular. His results have also been the subject of TV specials and hundreds of news stories, worldwide. In addition, we have published on molecular markers of chimpanzee species and subspecies (Publication 124) and on the relationships among the various subspecies (Publication 152). We have also used mitochondrial DNA sequences show diverse evolutionary histories of chimpanzees and other African hominoids. In Publication 184 we present the first family tree of the living African hominids that captures the real genetic diversity in each of the lineages. We completed the first comparison of species-level genetic diversity in gorillas, chimpanzees and humans using adequate samples (N = 60–800 per species) of apes of known geographic provenance. In a conclusion similar to those that characterized my work on invertebrates we found that, even among our closest living relatives, species are poorly or incorrectly defined. Compared to chimpanzees and gorillas, humans were shown to be unusually invariant genetically, a feature interpreted as reflecting different histories in the different clades. Our work has important taxonomic implications that are affecting on-going debates about the conservation management of the various isolated populations of great apes. Our reports have led to many other doctoral studies at other universities.

Publications

120. Morin, P.A. and D.S. Woodruff. Paternity exclusion using multiple hypervariable microsatellite loci amplified from nuclear DNA of hair cells. In: Paternity in Primates: Genetic Tests and Theories. Martin, R.D., A.F. Dixon and E.J. Wickings, eds. Basel, Karger, pp. 63–81. (1992a).
Note: First demonstration that questions regarding paternity and pedigree relations can be resolved using noninvasive genotyping based on microsatellite variation. Full collaboration between Woodruff and his graduate student, Phil Morin. [No. of citations: 44]

124. Morin, P.A., J. Moore and D.S. Woodruff. Identification of chimpanzee subspecies with DNA from hair and allele specific probes. Proceedings of the Royal Society of London B 249:293–297. (1992d).
Note: We discovered subspecies and species level DNA sequence differences at the cytochrome b locus and eliminated the need for sequencing by constructing ASO probes to facilitate rapid identification of animals of unknown geographic origin. Full collaboration between Woodruff & Morin. Jim Moore facilitated sample acquisition. [No. of citations: 38]

139. Morin, P.A., J. Wallis, J.J. Moore, R. Chakraborty and D.S. Woodruff. Non–invasive sampling and DNA amplification for paternity exclusion, community structure, and phylogeography in wild chimpanzees. Primates 34(3):347–356. (1993e).
Note: First detailed report of our Gombe chimpanzee study showing the wide range of issues that can be addressed using noninvasive genotyping of wild animals. Wallis (then Director of Gombe Reserve) and Moore (UCSD) facilitated and assisted with sample collection and interpretation. Chakraborty (Texas) contributed to the analysis. [No. of citations: 55]

146. Morin, P.A., J.J. Moore, R. Chakraborty, L. Jin, J. Goodall and D.S. Woodruff. Kin selection, social structure, gene flow and the evolution of chimpanzees. Science 265:1193–1201. (1994d).
Note: This is our major paper describing the results of the Gombe chimpanzee study. Provocative conclusions included the fact that West African chimpanzees may be specifically distinct from those in central and east Africa. Goodall and Moore (UCSD) facilitated and assisted with sample collection and the interpretation of the results. Chakraborty (Texas) contributed one statistical analysis performed by his student, Jin. [No. of citations: 401]

149. Morin, P.A., J. Wallis, J.J. Moore and D.S. Woodruff. Paternity exclusion in a community of wild chimpanzees using hypervariable simple sequence repeats. Molecular Ecology 3:469–478. (1994g).
Note: Detailed analysis of pedigree relations and social structure of the Kasakela social community of Gombe chimpanzees. Full collaboration between Woodruff and his graduate student, Morin. Wallis (then Director of Gombe Reserve) and Moore (UCSD) facilitated and assisted with sample collection and the interpretation of the results. [No. of citations: 120]

153. Morin, P.A., J.J. Moore and D.S. Woodruff. Chimpanzee kinship. Science 268:186–188. (1995b).
Note: Reply to the only criticism of Publication 145; namely, that a 1,000 km gap in our cross-Africa sampling precluded making judgment on the taxonomic status of the West African chimpanzees. We, and others, have subsequently filled this gap (see Paper 183).

165. Gagneux, P., D.S. Woodruff and C. Boesch. Furtive mating in female chimpanzees. Nature 387:358–359. (1997d).
Note: Our results showed that half of the offspring in the Tai Forest social community were sired by males not found in this community. Full collaboration between Woodruff, Boesch (Basel) and his student, Gagneux. Important: see publication 201. [No. of ISI citations: 93]

181. Varki, A., C. Wills, D. Perlmutter, D.S. Woodruff, F. Gage, J. Moore, K. Semendeferi, K. Benirschke, R. Katzman, R. Doolittle and T. Bullock. Great Ape Phenome Project. Science 282:239–240. (1998g).

182. Gagneux, P., C. Boesch and D.S. Woodruff. Female reproductive strategies, paternity and community structure in wild West African chimpanzees. Animal Behaviour 57:19–32. (1999a).
Note: Report with the detailed results excluded from the brief Nature note (Publication 164). [No. of citations: 79]

184. Gagneux, P., C. Wills, U. Gerloff, D. Tautz, P.A. Morin, C.B.B. Fruth, G. Hohmann, O. Ryder and D.S. Woodruff. Mitochondrial sequences show diverse evolutionary histories of African hominoids. Proceedings of the National Academy of Sciences, USA 96:5077–5082. (1999c).
Note: Major paper reporting for the first time the full extent of intra- and inter-specific variability in African hominoids based on individuals of known geographic origin. Results support our original finding that the West African chimpanzees are significantly different from the others. Full collaboration between Woodruff, Wills (UCSD) and graduate student, Gagneux. Others (U. Gerloff, D. Tautz, P.A. Morin, C.B.B. Fruth, G. Hohmann, O. Ryder) contributed unpublished sequence data. [No. of citations: 201]

185. Woodruff, D.S. Chimp cultural diversity. Science 285:836. (1999e).

191. McConkey, E.H., A. Varki, J. Allman, K. Benirschke, F. Crick, T.W. Deacon, F. de Waal, A. Dugaiczyk, P. Gagneux, M. Goodman, L.I. Grossman, D. Gumucio, T. Insel, K.K. Kidd, M.-C. King, K. Krauter, R. Kucherlapati, A. Motulsky G,, D. Nelson, P. Oefner, G. Palade, M. Ruvolo, O.A. Ryder, J. Sikela, C.-B. Stewart, A. Stone and D. Woodruff. A Primate Genome Project should be given high priority. Science 289:1295. (2000b).

201. Gagneux, P., D.S. Woodruff and C. Boesch. Furtive mating in female chimpanzees. Nature 414:508. (2001e).
Note: Linda Vigilant, in an independent analysis of relationships in the Tai Forest social community, found that 10 of 66 alleles and 9 of 33 individuals were incorrectly genotyped in Publication 165. Allelic dropout associated with the amplification of nuclear DNA from field-collected degraded hair samples and miscalled genotypes due to PCR artifacts (stutter bands) were the principal causes of scoring error. In this note we simply retract our previous paper. It was ironic as we had already presented one of the first analyses of the possible significance of this problem in genotyping from degraded, trace quantity templates (Publication 167), and former student Morin had independently provided a method of prescreening DNA extracts for more reliable genotyping (Mol. Evol. 10:1835). In Publication 209 (listed in Theme 5, above), I discuss the lessons learned. [No. of ISI citations: 5]

Dr. Phillip Morin collecting shed hair in a tree-top chimpanzee night nest, Gombe, Tanzania. He was the first to use hair as a DNA source for noninvasive genotyping of non-human primates and opened up the field of chimpanzee population genetics and phylogeography. (See Publications 118, 120, 124, 138, 139, 149, 155, 184, and especially 145.)

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Gibbons

I have published several reports on gibbon evolutionary and conservation genetics based on DNA sequences amplified from hair samples (Publications 130, 147, 151). In addition, the mitochondrial DNA control region sequences determined by a Masters student (Keri Monda 1995), a Chinese visiting scholar (Bing Su), and a German student (Phillip Kressirer) have been re-analysed using Baysian phylogenetic methods by Masters student Rachel Simmons (thesis: A Phylogenetic Study of the Gibbons (Hylobatidae) 2005; see manuscripts 215, 218, and C2). Our results were presented at the International Primatological Congress in Kyoto in 2010 (Manuscript C15).

Publications

130. Garza, J.C. and D.S. Woodruff. A phylogenetic study of the gibbons (Hylobates) using DNA obtained non–invasively from hair. Molecular Phylogenetics and Evolution 1(3):202–210. (1992j).
Note: Between 12 and 21 species and subspecies-level taxa of the Southeast Asian gibbons are recognized by different authorities and their phylogenetic relationships are poorly understood. Graduate student, Carlos Garza, and I used cytochrome b mtDNA sequences to provide this first genetic phylogeny. [No. of citations: 42]

147. Garza, J.C. and D.S. Woodruff. Crested gibbon (Hylobates (Nomascus)) identification using non-invasively obtained DNA. Zoo Biology 13:383–387. (1994e).
Note: Our genetic resolution of an identification problem involving captive gibbons was based on mtDNA sequence differences. Full collaboration between Woodruff and graduate student Carlos Garza. [No. of citations: 9.]

151. Woodruff, D.S. and R.L. Tilson. Genetic aspects of gibbon management in Thailand. In: Population and Habitat Viability Analysis Report for Thai Gibbons: Hylobates lar and H. pileatus. Tunhikorn, S. and 9. others, eds. IUCN/SSC Conservation Breeding Specialist Group, Apple Valley, MN, pp. 43–45. (1994h).
Note: Application of our genetic results to the management of captive and wild gibbons in Thailand. I provided data, interpretation and text for a workshop volume prepared by Tilson (CBSG).

215. Woodruff, D.S., K. Monda and R.E. Simmons. Mitochondrial DNA sequence variation and subspecific taxonomy in the white-handed gibbon, Hylobates lar. [Proceedings of the International Symposium on Southeast Asian Primate Research. Bangkok, October 17–20.] Natural History Journal of Chulalongkorn University Supplement 1:71–78. (2006a)
Note: Four allopatric subspecies are recognized in this species which ranges from China to Sumatra. Our survey of variation at a variable mtDNA locus (control region) in 60 apes in zoo collections revealed very little variation between the individuals studied. These results do not support the subspecific taxonomy but underscore the need to characterize animals of known geographic provenance.

220. Monda, K., R.E. Simmons, P. Kressirer, B. Su and D.S. Woodruff. Mitochondrial DNA sequence variation and phylogeny of the concolor gibbons, Nomascus. American Journal of Primatology 69: 1285–1306. (2007)
Note: provided the first comparative genetic characterization of all known species and revealed the probable phylogeny of the living taxa. [No. of citations: 4]

219. Su B, Kressirer P, Wang W, Jiang X, Wang YX, Woodruff DS, Monda K, Zhang YP. 1996. [Molecular phylogeny of Chinese Nomascus concolor concolor gibbons.] [in Chinese.] Science in China (C) 26:414–419.
Note: Publication discovered in 2007.

B14. Woodruff, D.S. Gibbon phylogeny still lies hidden in the trees: molecular genetic and chromosomal hypotheses. 23rd Congress of the International Primatological Society, September 2010, Kyoto, Japan.

C2. Simmons, R.E., K. Monda and D.S. Woodruff. Phylogenetics of gibbons (Hylobatidae): hypotheses based on mitochondrial and nuclear DNA sequences and chromosomal variation. Molecular Phylogenetics and Evolution in preparation.

C14. Woodruff, D.S. & Ashbacher, A. Ontogenetic color change in gibbons. American Journal of Primatology in preparation

C15. Woodruff, D.S. Gibbon phylogeny: molecular genetic and chromosomal hypotheses. Invited paper for: The Evolution of Gibbons: Molecular and Cognitive Evolution. C. Barelli, Hiro Hirai, U. H. Reichard, eds. (2012 proposed)

Carlos Garza demonstrating the two-handed gibbon DNA (hair) sampling method at Elephant Camp, near Chiang Mai, Thailand. Animals show are male and female sibs of the white-handed gibbon Hylobates lar. His studies of cytochrome b sequence level variation were used to explore phylogenetic relationships among gibbon species (Publications 130, 147, 151).

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Marmosets

A series of three papers describe the results of our study of marmoset evolution with postdoctoral fellows Nick Mundy and Caroline Nievergelt. Nievergelt’s formal genetic analysis of population structure and mating system in a long-watched wild population of the common marmoset in Brazil represents another “first” for my group (Publication 189).

Publications

178. Nievergelt, C.M., N.I. Mundy and D.S. Woodruff. Microsatellite primers for genotyping common marmosets (Callithrix jacchus) and other callitrichids. Molecular Ecology 7:1432–1434. (1998d).
Note: A methods paper, the first of a series on marmosets with postdoctoral fellows, Nievergelt and Mundy. [No. of citations: 20]

189. Nievergelt, C.M., L.J. Digby, U. Ramakrishnan and D.S. Woodruff. Genetic analysis of group composition and breeding system in a wild common marmoset (Callithrix jacchus) population. International Journal of Primatology 21(1):1–20. (2000f).
Note: Using microsatellite primers developed in Publication 177 we present the first analysis of population structure and mating system in a wild common marmoset population. This Brazilian marmoset community was originally studied by Digby who provided some blood samples and facilitated Nievergelt's field work. Woodruff's graduate student, Uma Ramakrishnan, contributed to the statistical analyses. [No. of citations: 64]

193. Mundy, N.I., A. Pissinatti and D.S. Woodruff. Multiple nuclear insertions of mitochondrial cytochrome b sequence in callitrichine primates. Molecular Biology and Evolution 17:1075–1080. (2000c).
Note: We report 4 derived nuclear sequences of mitochondrial origin. Such sequences confound phylogenetic reconstructions if unrecognized. A novel method of phylogenetic reconstruction, based on the large difference in rates of evolution at different codon positions among mitochondrial and nuclear clades, was used to determine whether the different nuclear paralogs represent independent transposition events or duplications following a single insertion. It was shown that at least three of the four nuclear clades represent independent transposition events. The insertion events giving rise to two of the nuclear clades predate the divergence of the callitrichines, whereas those leading to the other two nuclear clades may have occurred in the common ancestor of marmosets. A collaboration between Woodruff, postdoctoral fellow Mundy and our Brazilian collaborator Pissinatti. [No. of citations: 27]

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Lemurs

Postdoctoral fellow Caroline Nievergelt conducted a genetic characterization of the wild population of the endangered Aloatran gentle lemur, a subspecies restricted to the reed beds in a single lake in Madagascar. We have reported the results of our population genetic and phylogeographic studies in three papers.

Publications

195. Nievergelt, C.M., J. Pastorini and D.S. Woodruff. Genetic variability and phylogeography in the wild Aloatran gentle lemur population. In: Primatology and Anthropology: Into the Third Millennium. Soligo, C., G. Anzenberger and R.D. Martin, eds. Wiley, New York, pp. 175–179. (2002). Also published as Evolutionary Anthropology 11 (Supplement).
Note: Reviews phylogeographic aspects of Nievergelt’s genetic analyses. Zurich graduate student Pastorini contributed data on related taxa.

203. Nievergelt, C.N., T. Mutschler and D.S. Woodruff. Social system of the Alaotran gentle lemur (Hapalemur griseus alaotrensis): Genetic characterization of group composition and mating system. American Journal of Primatology 57:157–176. (2002b).
Note: First genetic characterization of the social structure and mating system of a specialized lemur restricted to the reed beds of a single lake. Postdoctoral fellow Caroline Nievergelt conducted the field work over a period of several years and carried out the genotyping in my laboratory. Mutschler was a Swiss student studying the ecology of this subspecies. [No. of citations: 28]

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Elephants

Genetic variation and evolution of elephants – I recruited two PhD students to conduct the first genetic survey of wild African and Asian elephants using DNA amplified from dung (protocols developed in my lab during 1997). This project constitutes an extension of the Thai small mammal study in that we up-scaled to megavertebrates of greater concern to conservation managers and the public.

Publications

188. Eggert, L.S. and D.S. Woodruff. Evolution and phylogeography of African forest elephants (Loxodonta africana cyclotis). Annual meeting of the Society for the Study of Evolution. Madison, WI, June 22–26, 1999. Program p. 26. (1999h).
Note: this report establishes our precedence over the claims of another worker to have been the first to genetically characterize the forest elephant.

190. Eggert, L.S. and D.S. Woodruff. The phylogeography and management units of the African forest elephant. 14th Annual Meeting, Society for Conservation Biology, Missoula, MT, June 9–12. Program and Abstracts, p. 160. (2000a).
Note: first phylogeographic analysis of forest elephants throughout their range.

197. Eggert, L.S., U. Ramakrishnan, N.I. Mundy and D.S. Woodruff. Polymorphic microsatellite DNA markers in the African elephant (Loxodonta africana) and their use in the Asian elephant (Elephas maximus). Molecular Ecology 9:2223–2225. (2000e).
Note: We report primer sequences for highly informative nuclear markers that permitted our studies of phylogeography, gene flow and genetic censusing of elephants (see Publications 205 and 210). Graduate student Eggert was assisted in the laboratory work by fellow student Ramakrishnan and former postdoctoral fellow Mundy. [No. of citations: 16]

205. Eggert, L.S., C.A. Rasner and D.S. Woodruff. The evolution and phylogeography of the African elephant (Loxodonta africana), inferred from mitochondrial DNA sequence and nuclear microsatellite markers. Proceedings of the Royal Society of London B 269:1993–2006. (2002d).
Note: This is the first report of the phylogeography of elephants across their entire range in Africa. We discovered that there are three genetically and geographically definable taxa of elephants in Africa: savanna elephants, forest elephants and west African elephants. Doctoral student Lori Eggert conducted the fieldwork and genotyping, with some lab assistance from Masters student Caylor Rasner. While this paper was in review with this journal in August 2001, Roca and O’Brien published a similar but less comprehensive study in Science. We withdrew our manuscript and revised it in the light of their report and it was finally published a year later. Our results are concordant with theirs but show how the savanna elephants are composed of two waves of forest elephant emigrants. Furthermore, we show that the west African elephants, which Roca did not sample, are also subspecifically or specifically distinct. Unlike Roca, who used traditional hunting and darting methods to obtain DNA samples, our results demonstrate that wild elephants can be genotyped noninvasively using DNA amplified from dung. [No. of citations: 70]

210. Eggert, L.S., J.A. Eggert and D.S. Woodruff. Estimating population sizes for elusive animals: the forest elephants of Kakum National Park, Ghana. Molecular Ecology 12:1389–1402. (2003d).
Note: This was the first demonstration that these large, dangerous, hard-to-see forest animals can be censused with noninvasive dung-based genotyping methods. Our estimate of population size (N = 223) was remarkably concordant with that reached using a traditional dung-counting method (N = 233) and also provided additional, previously unavailable, data on the sex of each individual and relationships among the animals present in a forest. Eggert collected the samples and performed the microsatellite genotyping; her husband assisted with the mathematics for an improved rarefaction analysis (which has subsequently been shown by another group to be more reliable than other methods). [No. of citations: 117]

Lori Eggert collecting DNA (dung) samples of west African elephants, Mole National Park, Ghana. Her surveys of mtDNA and nDNA variation in elephants across Africa led to the discovery of three taxa (species or subspecies) of living African elephants and, in particular, the discovery that the west African elephants were quite different from the savanna and forest elephants. This was reported in Publication 205. She also demonstrated that forest elephants could be censused (establishing both the number of elephants present and sex of each individual) noninvasively without being seen or directly encountered (Publication 210).

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